Les mécanismes de régulation post-transcriptionnelle sont des thématiques de recherche émergeantes en biologie moléculaire. La compréhension de ces régulations par les G- quadruplexes d’ARN de gènes dérégulés dans les maladies neurodégénératives pourrait donner des indices sur les mécanismes affectés lors de certaines maladies comme la maladie de Parkinson. Par ailleurs, les différents traitements ciblant certains G-quadruplexes sont à ce jour non spécifiques. Notre hypothèse est qu’il serait possible de moduler le niveau protéique de gènes impliqués dans la maladie de Parkinson, et ce par la modulation de la liaison entre des protéines et des G-quadruplexes situés dans des régions 5’UTR. L’objectif générale de ce projet est l’étude de protéines liant les G-quadruplexes situés dans les régions 5’UTR de gènes impliqués dans la maladie de Parkinson. Pour accomplir ce travail, nous proposons comme objectifs spécifiques d’:
• Identifier et étudier des G-quadruplexes d’ARN impliqués dans des gènes associés à la maladie de Parkinson;
• Identifier des protéines étant en mesure de lier ces G-quadruplexes; et,
• Caractériser l’interaction entre les protéines et les G-quadruplexes d’ARN trouvés; et de,
• Moduler la formation du complexe protéine-G4.
Ces objectifs nous permettrons de mieux comprendre l’importante des G-quadruplexes au sein de la régulation post-transcriptionnelle.
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2R
ÉSULTATSArticle « Guanine Nucleotide-Binding Protein-Like 1 (GNL1)
binds RNA G-quadruplex structures in genes associated with
Parkinson’s disease »
Auteurs de l’article: Marc-Antoine Turcotte, Jean-Michel Garant, Hélène Cossette- Roberge et Jean-Pierre Perreault
Statut de l’article: Soumis à Journal of RNA biology le 20 Août 2020
Avant-propos: Marc-Antoine Turcotte a écrit la totalité de l’article et celui-ci a été révisé et corrigé par le directeur de recherche, Jean-Pierre Perreault. Marc-Antoine Turcotte a réalisé la presque totalité des expériences, mis à part les analyses sur G4 RNA Screener (Jean- Michel Garant) et les expériences de fluorescence au NMM sur les 11 pG4 (Hélène Cossette- Roberge). Le projet de recherche s’est échelonné sur les deux ans de maîtrise.
Résumé :
Au niveau post-transcriptionnel, les ARN sont hautement régulés dans les maladies neurodégénératives et la présence de quelques mutations peut affecter le sort des cellules neuronales. À ce jour, l'impact de la régulation des G-quadruplexes (G4) dans certaines maladies neurodégénératives, comme la maladie de Parkinson (PD), n'a jamais été analysé. Dans cette étude, nous avons initialement identifié in silico des G4 potentiels (pG4) présents dans les gènes dérégulés liés au système nerveux et montré qu’il y existait un enrichissement significatif en pG4. Ensuite, il a été démontré par des tests biochimiques que plusieurs des séquences G4 récupérées dans la région 5’ non traduite (5’UTR) d’ARNm associés à la maladie de Parkinson se repliaient en G4 in vitro. Le clonage de la longueur totale du 5’UTR de ces candidats en amont d’un système rapporteur de luciférase a permis de montrer que le G4 de la protéine ubiquitine ligase Parkin RBR E3 (PRKN) et de la protéine 35 associée aux protéines vacuolaires sortantes (VPS35) réprimait de manière significative la traduction des deux gènes dans les cellules SH-SY5Y. Par la suite, une stratégie de purification par affinité en utilisant l'une ou l'autre de ces deux séquences G4 comme appâts a isolé la protéine Guanine Nucleotide-Binding Protein-Like 1 (GNL1). Cette dernière s'est avérée avoir une affinité plus élevée pour les séquences sauvages G4 que mutantes. Cette étude met en lumière un nouveau mécanisme moléculaire important pour la maladie de Parkinson et propose de nouvelles avenues pour moduler la traduction des maladies.
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Guanine Nucleotide-Binding Protein-Like 1 (GNL1) binds RNA G-
Quadruplex structures in genes associated with Parkinson’s disease
Marc-Antoine Turcotte, Jean-Michel Garant, Hélène Cossette-Roberge, and Jean-Pierre Perreault*
Department of Biochemistry, Pavillon de recherche appliquée sur le cancer, Université de Sherbrooke, Sherbrooke, Québec, Canada, J1E 4K8
*To whom correspondence should be addressed: Email: jean-pierre.perreault@usherbrooke.ca Telephone Number: 819-821-8283
Fax Number: 819-564-5340
ORCID ID: 0000-0002-4559-5541
Running Title: RNA G4s as translational regulators
Key words: G-quadruplex, translation, RNA binding proteins, transcriptome, neurosciences
List of abbreviations: CDS, Coding sequence; EMSA, electrophoretic mobility shift assay; G4, G-Quadruplex; GNL1, Guanine Nucleotide-Binding Protein-Like 1; HGNC, HUGO Gene Nomenclature Committee; Kd, dissociation constant; KEGG, Kyoto Encyclopedia of Genes and Genomes; ND, Neurodegenerative diseases; NMM, N-Methyl Mesoporphirine; PD, Parkinson’s disease; pG4, Potential G4; PRKN, Parkin RBR E3 Ubiquitin Protein Ligase; UTR, Untranslated region; VPS35, Vacuolar Protein Sorting-Associated Protein 35
ABSTRACT
RNAs are highly regulated at the post-transcriptional level in neurodegenerative diseases and just a few mutations can significantly affect the fate of neuronal cells. To date, the impact of G-quadruplex (G4) regulation in neurodegenerative diseases like Parkinson's disease (PD) has not been analyzed. In this study, in silico potential G4s located in deregulated genes related to the nervous system were initially identified and were found to be significantly enriched. Several G4 sequences found in the 5’ untranslated regions (5’UTR) of mRNAs associated with Parkinson's disease were demonstrated to in fact fold in vitro by biochemical assays. Subcloning of the full-length 5’UTRs of these candidates upstream of a luciferase reporter system led to the demonstration that the G4s of both Parkin RBR E3 Ubiquitin Protein Ligase (PRKN) and Vacuolar Protein Sorting-Associated Protein 35 (VPS35) significantly repressed the translation of both genes in SH-SY5Y cells. Subsequently, a strategy of using label-free RNA affinity purification assays with either of these two G4 sequences as bait isolated the Guanine Nucleotide-Binding Protein-Like 1 (GNL1). The latter was shown to have a higher affinity for the G4 sequences than for their mutated version. This study sheds light on a new molecular mechanism important in Parkinson's disease, and proposes new avenues for modulating disease.
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INTRODUCTION
G-quadruplexes (G4) are non-canonical secondary structures found in both DNA and RNA molecules. In cells, as well as in vitro, four guanine residues can be linked together by Hoogsteen bonds in order to form a structure called a G-quartet that can be stabilized by the presence of a monovalent cation, usually potassium (Rouleau et al. 2017b). Canonical G4 structures respect the motif G3-N1-7-G3-N1-7-G3-N1-7-G3 (where N indicates A, U, C or G). However, some studies have shown that non-canonical G4s may have longer loops, only two quartets, or the presence of bulges in G-runs (Bolduc et al. 2016; Jodoin et al. 2014; Xiao et al. 2018). While DNA G4s can be present in many conformations, RNA G4s, which are more stable than their DNA counterparts, are restricted to folding into parallel conformations (Małgowska 2016).
Studies on G4s have demonstrated that they exhibit many regulatory functions in cells. For example, DNA G4s have been demonstrated to be implicated in DNA replication, transcription and in telomere elongation (Kim, 2019; Lerner et Sale, 2019; Neidle et Parkinson, 2003). In mRNA, G4s have been located in the 5’ untranslated region (UTR), the coding sequence (CDS) and the 3’UTR (Rouleau et al. 2017b). G4s located in the 5’UTR have been shown to impair ribosome scanning, leading to repression of the cap-dependent translation (Beaudoin et Perreault, 2010; Bugaut et Balasubramanian, 2012). However, some G4s have been also demonstrated to enhance cap-independent translation by the stabilization of internal ribosome entry sites (Jodoin et al. 2019). On the other hand, CDS G4s have been shown to impair translational elongation and to induce frameshifting (Endoh et al. 2013; Yu et al. 2014). Finally, G4s located in the 3’UTR have been shown to be associated with both translational repression and the regulation of polyadenylation (Arora et Suess, 2011; Beaudoin et Perreault, 2013). Furthermore, G4s were found to have an impact on other types
of RNAs, like pre-miRNA, where they have been shown to regulate RNA processing (Rouleau et al. 2017a). Lastly, in long non-coding RNA, they often sequestrate G4 RNA binding proteins (Matsumura et al. 2017).
It has been proposed that RNA G4s are globally unfolded in eukaryotes (Guo et Bartel, 2016). Since they are known to be relatively stable structures, intracellular mechanisms must regulate their formation. Yet only a few proteins have been shown to be able to fold, unfold or stabilize G4s (Mendoza et al. 2016). Examples of these RNA binding proteins are the G-Rich RNA Sequence Binding Factor 1 (GRSF1), a protein known to unfold RNA G4s and to facilitate degradosome-mediated decay, and nucleolin, a protein known to stabilize RNA G4s (Masuzawa et Oyoshi, 2020; Pietras et al., 2018). Since each G4 function is thought to be regulated by specific binding proteins, targeting them appears to be an attractive avenue for controlling the associated post-translational events (Sun et al. 2019).
Over the years, several G4 motifs have been associated with important mechanisms in neurological diseases (Ishiguro et al., 2016; Koukouraki et Doxakis, 2016). For example, the RNA foci C9orf72, which forms multiple repeats of G4s, has been linked to both frontotemporal dementia and amyotrophic lateral sclerosis (Schludi et Edbauer, 2018). A mutated miRNA associated with Alzheimer’s disease has been demonstrated to fold into a G4 (Imperatore et al. 2020). In fragile X syndrome, fragile X mental retardation protein, a protein known to both bind to mRNAs containing a G4 structure and to regulate their transport, was shown to be less abundant. This reduction also directly affects the translation of several other mRNAs (Melko et Bardoni, 2010). In the present study, the original identification of the Guanine Nucleotide-Binding protein-like 1 (GLN1) as an RNA binding protein (RPB) that binds to the G4 structures located in the 5’UTRs of both VPS35 and PRKN, two genes that are deregulated in Parkinson's disease, is reported.
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MATERIALS AND METHODS Bioinformatic analyses
In order to retrieve genes associated with nervous system diseases, BRITE hierarchy files (br08402) were downloaded from the disease data-oriented entry points of the Kyoto Encyclopedia of Genes and Genomes (KEGG) (Kanehisa et al. 2015). The gene symbols from each line associated with the nervous system were conserved and then transformed into a list file. Next, the symbols were corrected using the Multi-symbol checker of the HUGO Gene Nomenclature Committee (HGNC) (Braschi et al. 2019). Then, the RNA.fa.gez files were downloaded from Refseq (NCBI, March 2018), and each gene from the HGNC list was associated with a Fasta transcript sequence. The Fasta files were then submitted to the G4 RNA Screener software using the default parameters, and all windows with a G4NN score >0.5 were considered as being positive (Garant et al. 2017). Finally, pG4 windows were annotated by their transcript position (5’UTR, CDS and 3’UTR) using the RefSeq CDS annotation "ncbiRefSeqCds.txt.gz "(May 2018).
For the purpose of comparing the KEGG results to other databases, tab-separated values files were retrieved from the Human Protein Atlas for the enriched neuronal genes. They were then associated with a Fasta transcript file and submitted to the G4 RNA screener. Genes with a G4NN score >0.5 were considered as being positive.
Design of the oligonucleotides
Oligonucleotides corresponding to the potential G4 sequences were designed to include both upstream and downstream wild type flanking sequences of up to 20 to 25 nucleotides (nts) in length. G/A mutants (several Gs substituted by As) were also synthesized for each WT G4 sequence (see in Tables S1 and S2 for sequence details). When required for in vitro
transcription, a T7 promoter sequence (5’-TAATACGACTCACTATAG1-3-3’, where G1-3 indicates a stretch of 1 to 3 consecutive guanines) was added to the sense strand. All oligonucleotides were purchased from Biobasic.
In vitro transcription
In order to reconstitute the full-length WT G4 and G/A mutant sequences (which are70-90 nts in size), DNA templates were synthesized via a PCR filling strategy using both forward and reverse primers at a concentration of 2 mM. Then, purified Pfu DNA polymerase (2 µL) was added to a final volume of 100 µL containing 2 mM MgSO4, 0,2 mM dNTPs, 10 mM (NH4)2SO4, 10 mM KCl, 20 mM Tris-HCl (pH 8.8), 0,1 % Triton-X-100 and 5 % DMSO. The PCR program used was 12 cycles of 95°C (1 min), 54°C (1 min) and 72°C (1 min), and was terminated with a final elongation of 5 min at 72°C. The DNA was then ethanol precipitated and dissolved in 10 µL of ultrapure water. DNA integrity and size were then verified by 2% agarose gel electrophoresis.
In order to synthesize RNA in vitro, 100 µL reactions containing 5 µL of the PCR filling DNA reactions, 2 µL of purified T7 RNA polymerase, 0.01 U of pyrophosphatase (Roche Diagnostics) and final concentrations of 80 mM HEPES-KOH (pH 7.5), 24 mM MgCl2, 5 mM NTPs, 2 mM spermidine and 40 mM DTT were prepared and then were incubated at 37°C for 3 h. After this incubation, a DNase treatment (5U, Promega) was performed for 20 min at 37°C., The proteins were then removed by phenol/chloroform extraction, and the RNA ethanol precipitated. The RNA samples were dissolved in 30 µL ultrapure water, mixed with 60 µL of loading buffer (94 % formamide, 0.1 % EDTA and 0.025% w/v each of bromophenol blue and xylene cyanol) and were then purified using denaturing (8 M urea) 5% polyacrylamide gels (19:1). The RNA bands of the appropriate
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sizes were cutted and nucleic acids eluted overnight in a solution that contained 1 mM EDTA, 0.1 % SDS and 0.5 M ammonium acetate, ethanol precipitated, dried and dissolved in 100 µL of ultrapure water. For the transcriptions of both pMA-RQ VPS35-4xS1m and pMA-RQ PRKN-4xS1m, the plasmids were digested with EcoR1 and HindIII prior to the transcription reaction.
N-Methyl Mesoporphyrin (NMM) fluorescent assays
With the aim of probing G4 formation, fluorescence assays using NMM were performed as described previously (Jodoin et Perreault, 2018). Briefly, either the WT or a G/A mutant G4 RNA (200 pmol) was added to folding buffer (20 mM Li-cacodylate (pH 7.5) and 100 mM of either LiCl or KCl) in a final volume of 50 µL. The reactions were then heated to 70°C and allowed to slowly cool for 1 h at room temperature. Next, 50 µL of 2X in-line probing buffer (40 mM Li-cacodylate (pH 8.5), 40 mM MgCl2 and 200 mM of either LiCl or KCl) were added, yielding a final volume of 100 µL. The NMM ligand (1 µL at a concentration of 0.5 mM; N-Methyl-Mesoporphyrin IX, NMM580, Frontier Scientific Inc., Logan, Utah) was then added and the reaction incubated for 30 min at room temperature while being protected from light. The fluorescence intensity was monitored using an Hitachi F-2500 fluorescence spectrophotometer with an excitation wavelength of 399 nm and the emission wavelength being measured between 500 nm and 650 nm in a 10 mm quartz cuvette. The fluorescence at 605 nm was used for quantification. All NMM assays were performed at least in duplicate.
Dephosphorylation and radioactive labeling
The Antarctic Phosphatase kit (New England Biolabs) was used to prepare radioactive transcripts by incubating the RNA samples (50 pmol) at 37°C for 45 min. Upon completion
of the reactions, the phosphatase was inactivated at 70°C for 5 min and the reaction was then immediately chilled on ice. A fraction of the dephosphorylated RNA (10 pmol) was then incubated at 37°C for 1 h with the T4 Polynucleotide Kinase (PNK) kit (Promega) in the presence of [γ32P]ATP (2 μL; 6 000 Ci (222 TBq)/mmol in 50 mM Tricine (pH 7.6), PerkinElmer). Two volumes of loading buffer (94 % formamide, 0.1 % EDTA and 0.025% (w/v) each of bromophenol blue and xylene cyanol) were then added and the resulting samples were then electrophoresed through denaturing (8 M urea) 5% polyacrylamide gels (19:1). The bands of appropriated sizes were cut out of the gels, and the transcripts were then eluted overnight in a solution containing 1 mM EDTA, 0.1 % SDS and 0.5 M ammonium acetate. The eluted transcripts were then ethanol precipitated and th resulting pellets dissolved in 20 μL of ultrapure water.
In-line probing
In order to confirm G4 folding, in-line probing was performed as described previously (Beaudoin et al., 2013; Jodoin et Perreault, 2018). Briefly, either WT or G/A mutant G4 RNA labeled with 32P (50 000 counts per minute (CPM) per reaction) was incubated in folding buffer (20 mM Li-Cacodylate (pH 7.5) and 100 mM of either LiCl or KCl) in a final volume of 10 µL for 5 min at 70°C before being slow cooled for 1 h at room temperature. Subsequently, 50 µL of 2X in-line buffer (40 mM Li-Cacodylate (pH 8.5), 40 mM MgCl2 and 200 mM of either LiCl or KCl) and 40 µL of ultrapure water were added to the mixtures before incubating them for 40 h at room temperature. The RNAs were then ethanol precipitated in the presence of glycogen, ethanol washed, dried and dissolved in 20 µL of loading buffer (94 % formamide, 0.1 % EDTA and 0.025% (w/v) of xylene cyanol). The nucleotide ladder was prepared by incubating 25 000 CPM of 32P-labeled RNA for 1 min at
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room temperature with 1 µL of 2 N NaOH. The reactions were then neutralized by adding 3 µL of 1 M Tris-HCl (pH 7.5), and the RNA ethanol precipitated in the presence of glycogen before being dissolved in 20 µL of loading buffer (94 % formamide, 0.1 % EDTA and 0.025% (w/v) of xylene cyanol). For the RNase T1 ladder, both WT and G/A mutant G4 RNAs were incubated for 2 min at 37 °C with 0.6 U of T1 RNase (Roche Diagnostic) in 20 mM Tris-HCl (pH 7.5), 10 mM MgCl2 and 100 mM LiCl buffer. The reactions were then stopped by the addition of 20 µL of formamide dye loading buffer which contained only xylene cyanol as colourant. Equal amounts of CPMs for each condition and ladder were used, with the exception of the T1 ladder for which three times less CPM were used. The reactions were then adjusted to 10 µL by addition of formamide loading dye buffer and then electrophoresed through denaturing (8 M urea) 10% polyacrylamide gels. The resulting gels were dried for 50 min at 60°C and were then exposed to a phosphoscreen for 16 h. The results were visualized using a Typhoon Trio imaging system (GE Healthcare), and the band quantification was performed using the SAFA semi-automated software (Laederach et al. 2008). The hydrolysis ratio for each nucleotide position is represented by the band intensity in the presence of KCl divided by that in the presence of LiCl. All experiments were performed at least in duplicate.
Cell cultures
SH-SY5Y cells (ATCC, CRL-2266) were cultivated in Dulbecco’s modified Eagle medium/F12 with Glutamax (Gibco) supplemented with 10% fetal bovine serum (FBS; Wisent). Human Kidney Embryo 293T (HEK293T) cells (ATCC, CRL-3216) were cultivated in DMEM (Multicell) supplemented with 10 % FBS. When the cells reached a
confluence of 80-90 % they were subcultured at dilutions of 1:2 and 1:20, respectively. All cultures were incubated at 37°C with a 5 % CO2 atmosphere.
Molecular cloning
The PsiCHECK-2 plasmids used in the luciferase assays were prepared as follows. Puc57 plasmids containing either the full-length WT or G/A mutant 5’UTR G4 sequences of VPS35, LRRK2, SNCA and PRKN were purchased from Biobasics (see Table S1 for the full sequences). The 5’UTR sequences were codigested with SpeI and SalI, and were then ligated upstream of the Renilla luciferase in a modified version of the PsiCHECK-2 plasmid (Figure S2) that had previously been digested with the same restriction enzymes. The insertion was then confirmed by PCR, and the plasmids were sequenced at the Plateforme de Séquençage of Université Laval.
The PsiCHECK-2 plasmids prepared previously were used as templates in PCR reactions in order to generate G4 regions containing EcoR1 and BamH1 restriction sites located in 5’ and 3’, respectively. The resulting DNA templates were codigested with EcoRI and BamHI and ligated into pMA-RQ plasmid containing the 4xS1m aptamer (Leppek et Stoecklin, 2014). The insertion was confirmed by PCR, and all constructs were sequenced at the Plateforme de Séquençage of Université Laval.
Luciferase assay
SH-SY5Y and HEK293 cells were subcultured at densities of 80 000 and 22 500 cells per cm2 24 h, respectively, before transfection. They were then transfected in triplicate with Lipofectamine 2000 as described by the manufacturer (Thermo Fisher Scientific) using 500 ng of pUC19 and 50 ng of PsiCHECK-2 in each well of 24 well plates. A day later, the
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medium was removed and the cells were washed once with PBS. The cells were then dislodged and suspended by scraping in the presence of 500 µL of PBS. Four hundred microliters of the resulting suspension were then used to extract proteins, while the remaining 100 µL were used to extract total RNA. Both fractions were centrifuged at 4°C for 10 min at 2400 x g and the supernatants removed. The cell pellets for RNA isolation were frozen at - 80°C until treated, while 100 µL of protein lysis buffer (Dual-luciferase reporter assay system, Promega) was added to the cell pellets destined for protein extraction. In order to lyse the cells, the solutions were then incubated for 15 min at room temperature. Only a fraction (5 µL) of the resulting solution was then used for the luciferase assays as suggested by the manufacturer’s protocol. Luminescence was then monitored on a Glomax 20/20 luminometer with an integration time of 5 sec. The ratios of the Renilla/Firefly luciferases were then calculated and presented as the means and standard deviation of 3 experiments.
RNAs were extracted with Trizol according to the manufacturer's protocol (Thermo Fisher Scientific). When required, the RNAs were quantified by digital RT-PCR at the Laboratoire de Génomique Fonctionnel de Université de Sherbrooke.
G4 ligands
SH-SY5Y cells at a confluence of 70% were incubated for 24 h with either 10 µM of BRACO19 or 5 µM of Phen-DC3. Ultrapure water was used as negative control for BRACO19 and DMSO for Phen-DC3. After the 24 h were complete, the cells were washed once with 1X PBS and were then harvested by centrifugation for 5 min at 600 x g. The resulting supernatants were discarded and the cells were incubated with 20 µL of KCl-lysis buffer (50 mM Tris-HCl (pH 7.5), 100 mM KCl, 50 mM NaCl, 1 mM DTT, 0,1 % Triton X- 100, 10 % glycerol and 1 mM MgCl2) for 30 min at 4°C. The cellular lysates were then
centrifuged at 17 000 x g for 20 min at 4°C, and the resulting supernatants were kept. The